This proposal entitled "Dynamic Regulation of Shank3 and ASD" examines molecular mechanisms that link mutations of Shank3 with Autism Spectrum Disorder. Mutations of the open reading frame of Shank3 have been identified in multiple cases of ASD, however, it is not yet known how these mutations produce disease. Shank3 is a scaffolding protein that interacts with metabotropic glutamate receptors, AMPA type glutamate receptors and the NMDA receptor associated proteins, and appears to play a role in coordinating physiological interactions between these receptors at excitatory synapses. Shank3 also interacts with Homer, and mutations that disrupt Homer binding are associated with the most severe forms of ASD. To explore the molecular basis of ASD, we have generated a conditional KI of Shank3 that mimics human ASD in that the mutant protein lacks the C-terminus and the Homer binding site. Aim 1 examines this Shank3 KI mouse as a model of ASD using a combination of behavioral, biochemical and physiological approaches. These studies will explore the hypothesis that mutant Shank3 acts by a "gain of function" mechanism, either to alter signaling at the synapse or to accelerate the degradation of associated proteins including Shank3 expressed by the Wt allele. The latter hypothesis is based on preliminary findings that the expression of Shank3 is critically dependent on Homer binding. Aim 2 will examine the molecular mechanism of Shank3 degradation and explore the role of Deep orange (Dor). Dor is a member of the Vacuolar protein sorting (VPS) complex that functions with both proteosomes and lysosomes, and physically associates with Shank3 in a manner that is inhibited by Homer. Studies will utilize a conditional KO of Dor to test the prediction that Dor controls the level of Shank3 at synapses and trans heterozygote analysis will test if genetic knock down of Dor can rescue deficits due to Shank3 mutation. These studies examine novel mechanisms in synaptic biology and may provide new insights into how mutation of Shank3 leads to ASD. PUBLIC HEALTH RELEVANCE: This proposal relates to Recovery Act Limited Competition: Research to Address the Heterogeneity in Autism Spectrum Disorders (R01) targeted to Model Development for Understanding Neurobiological Mechanisms and Behavioral Model Development. Studies will examine two new mouse genetic models to evaluate how mutations of Shank3 alter synaptic function and behavior.